Amino sample preparation

The nitration of 6-methoxyquinoxaline in concentrated sulfuric acid at 0°C gives 6-methoxy-5-nitroquinoxaline. The position of the nitro group is confirmed by reduction of the product to 5-amino-6-methoxy-quinoxaline identical with a sample prepared from 2,3,4-triamino-anisole and glyoxal ... 

Sulfoxides without amino or carboxyl groups have also been resolved. Compound 3 was separated into enantiomers via salt formation between the phosphonic acid group and quinine . Separation of these diastereomeric salts was achieved by fractional crystallization from acetone. Upon passage through an acidic ion exchange column, each salt was converted to the free acid 3. Finally, the tetra-ammonium salt of each enantiomer of 3 was methylated with methyl iodide to give sulfoxide 4. The levorotatory enantiomer was shown to be completely optically pure by the use of chiral shift reagents and by comparison with a sample prepared by stereospecific synthesis (see Section II.B.l). The dextrorotatory enantiomer was found to be 70% optically pure. 

A general feature of optimum sample preparation is that maximum recovery of the analyte is observed. Consider a graph of recovery vs. variation in one experimental condition. Figure 5 shows such a graph, with temperature as the experimental variable. The curve exhibits a maximum and a decline on either side of the maximum. The assay will be most reproducible at the point of zero slope, i.e., at the maximum recovery, because small variations in conditions will not affect the result. In hydrolysis of a protein to its constituent amino acids, for example, it will be found that at very high temperatures or long hydrolysis times, degradation of the product amino acids occurs, while at low temperatures or short hydrolysis times, the protein... 

With the increased popularity of LC-MS, the problem of overlapping enantiomer peaks from other amino acids has largely been resolved. The mass spectrometer can act as an additional dimension of separation (based on mass to charge ratio). Thus, only amino acids having the same mass-to-charge ratio must be separated achirally (see Desai and Armstrong, 2004). This additional dimension of separation also has implications for the applications in the matrices discussed previously. With the ability of the mass spectrometer to discriminate on the basis of mass, this lessens the need for complete achiral separation. For example, an LC-MS method was recently developed to study the pharmacokinetics of theanine enantiomers in rat plasma and urine without an achiral separation before the enantiomeric separation (Desai et al., 2005). In such matrices, proteins must still be removed by appropriate sample preparation. 

PITC has been used extensively in the sequencing of peptides and proteins and reactions under alkaline conditions with both primary and secondary amino acids. The methods of sample preparation and derivatization follow a stringent procedure which involves many labour-intensive stages. However, the resulting phenylthio-carbamyl-amino acids (PTC-AA s) are very stable, and the timing of the derivatization step is not as critical as when using OPA. 

The Western blot method is often used in the analysis of host cell impurities. It can be used to identify a recurring impurity. O Keefe et al. used a Western blot to identify an E. coli protein impurity in the preparation of the recombinant fibroblast growth factor (aFGF).29 By using specific antisera to the E. coli host cell proteins, they were able to isolate the impurity and determine its N-terminus amino acid sequence to confirm its identity. Antibodies could be used to determine the concentration of this impurity in sample preparations. 

The applicability of cinchonan carbamate CSPs for bioanalytical investigations using HPLC-ESI-MS/MS has been demonstrated by Fakt et al. [120]. The goal was the stereoselective bioanalysis of (R)-3-amino-2-fluoropropylphosphinic acid, a y-aminobutyric acid (GABA) receptor agonist, in blood plasma in order to determine whether this active enantiomer is in vivo converted to the 5-enantiomer. In this enantioselective HPLC-MS/MS bioassay, sample preparation consisted of... 

A third type of detector is the intrinsic or native fluorescence detector that utilizes native fluorescence properties of amino acids. The sensitivity of this detector is between UV/PDA and LIF detection. The advantage of this technique over pre-labeling is that there is no pre-labeling step required therefore, the sample preparation is relatively simple, and the sensitivity is improved over UV/LIF. However, the intrinsic fluorescence detection relies on the presence of Tryptophan (Try), Tyrosine (Tyr), Phenylalanine (Phe), and this detector has just become commercially available. 

Sample preparation for HPLC analysis of free amino acids usually involves an extraction step (for solid matrices), followed by purification to remove possible impurities. When the aim of the... 

The most important point during sample preparation is to prevent oxidation of ascorbic acid. Indeed, it is easily oxidized by an alkaline pH, heavy metal ions (Cu and Fe ), the presence of halogens compounds, and hydrogen peroxide. The most suitable solvent for this purpose is metaphosphoric acid, which inhibits L-ascorbic oxidase and metal catalysis, and it causes the precipitation of proteins. However, it can cause serious analytical interactions with silica-based column, e.g., C18 or amino bonded-phases [542] and it could co-elute with AA. 

The vast differences in the functional groups possessed by the side chains allows for their chromatographic separation. However, closely related compounds can still be tough to resolve. For example, leucine and isoleucine tend to afford very tight resolution. The chemical differences in the side chains can be very problematic when it comes to sample preparation. This is especially true for the acid hydrolysis of proteins to liberate their constituent amino acids in the free form. The different functional groups exhibit different chemical stabilities in an acid environment. 

There are two major categories for amino acid analysis (a) free amino acid analysis and (b) determination of total amino acid content. The total amino acid content includes contributions from the free amino acids and the amino acids that are originally protein bound. These protein-bound amino acids must first be liberated before chromatographic analysis. This necessitates a more extensive, and problematic, sample preparation. Because the sample preparation procedures are so disparate, it is convenient to address these two categories of amino acid analyses separately. It should be noted that while the sample preparations for these analyses are quite different, both utilize essentially the same chromatographic techniques for the second stage of amino acid analysis. 

The sample preparation for analysis of free amino acids generally involves two steps. The first step is to ensure complete dissolution of the target amino acids. For beverages, this might al-... 

Sulfosalicylic acid has most commonly been used to precipitate proteins prior to ion-exchange amino acid analysis (11). In this mode, SSA allows for a very simple sample preparation that requires only centrifugation of the precipitated sample and then direct injection of the resulting supernatant solution. The supernatant solution is already at an appropriate pH for direct injection. Also, the SSA does not interfere chromatographically since it elutes essentially in the void volume of the column. It has been noted that, if an excessive amount of SSA is employed, resolution of the serine/threonine critical pair can suffer (12). The use of SSA prior to reversed-phase HPLC can be more problematic, since its presence can interfere with precolumn deriva-tization. For example, Cohen and Strydom (13) recommend the separation of the amino acids from the SSA solution on a cation-exchange resin prior to derivatization with phenylisothiocya-nate (PITC). 

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